Tom M. Ramseier

The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli

The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator‐containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR‐binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.

Cooperative Interaction Between Cra and Fnr in the Regulation of the cydAB Operon of Escherichia coli

In vivo and in vitro experiments are reported demonstrating that the catabolite repressoractivator (Cra) protein (formerly designated FruR) regulates expression of the cydAB operon of Escherichia coli encoding cytochrome d oxidase. The Fnr protein is required for Cra-mediated transcriptional control, but the ArcA protein antagonizes the response to Cra. The results establish that Fnr, ArcA, and Cra exert their effects in an interdependent fashion.

cAMP-cAMP receptor protein complex: five binding sites in the control region of the Escherichia coli mannitol operon.

The control region of the mannitol (mtl) operon of Escherichia coli has been shown to contain five cAMP receptor protein (CRP) binding sequences, the most yet reported for any operon. A DNA fragment encompassing the entire mtl operon regulatory region was generated by PCR, and the binding of the cAMP-CRP complex was studied. Using restrictional analysis to separate, delineate and destroy the various putative CRP binding sites, all five sites were shown to be functional for CRP binding in vitro. Four of these sites bound the cAMP-CRP complex with high affinity while the fifth site (the most distal relative to the transcriptional start site) bound the complex with lower affinity. Simultaneous binding of cAMP-CRP complexes to several of these sites was demonstrated. The results serve to identify and define five dissimilar CRP binding sites in a single operon of E. coli. A model for mtl operon transcriptional initiation and repression complexes is presented.